Chemotherapy-induced pyroptosis is mediated by BAK/BAX-caspase-3-GSDME pathway and inhibited by 2-bromopalmitate

Abstract

Many chemotherapy treatments induce apoptosis or pyroptosis through BAK/BAX-dependent mitochondrial pathway. BAK/BAX activation causes the mitochondrial outer membrane permeabilization (MOMP), which induces the activation of pro-apoptotic caspase cascade. GSDME cleavage by the pro-apoptotic caspases determines whether chemotherapy drug treatments induce apoptosis or pyroptosis, however, its regulation mechanisms are not clear. In this study, we showed that TNFα+CHX and navitoclax-induced cancer cell pyroptosis through a BAK/BAX-caspase-3-GSDME signaling pathway. GSDME knockdown inhibited the pyroptosis, suggesting the essential role of GSDME in this process. Interestingly, GSDME was found to be palmitoylated on its C-terminal (GSDME-C) during chemotherapy-induced pyroptosis, while 2-bromopalmitate (2-BP) could inhibit the GSDME-C palmitoylation and chemotherapy-induced pyroptosis. Mutation of palmitoylation sites on GSDME also diminished the pyroptosis induced by chemotherapy drugs. Moreover, 2-BP treatment increased the interaction between GSDME-C and GSDME-N, providing a potential mechanism of this function. Further studies indicated several ZDHHC proteins including ZDHHC-2,7,11,15 could interact with and palmitoylate GSDME. Our findings offered new targets to achieve the transformation between chemotherapy-induced pyroptosis and apoptosis.

Fig. 1. BAK/BAX deletion inhibits TNFα+CHX or navitoclax-induced pyroptosis.

a Expresssion level of GSDMD and GSDME in various cell lines. b Expression of BAK/BAX in wild type (WT) and BAK/BAX double knockout (DKO) HCT116 cells. c, d At the indicated time points, the percentage of LDH release in the culture supernatants from HCT116 WT and DKO was measured after TNFα+CHX (c) or navitoclax (d) treatment. Error bars in this and subsequent figures: mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. e, f At the indicated time points, immunoblottings of GSDME, caspases, and cleaved caspases were performed in HCT116 WT and DKO cells treated with TNFα+CHX (e) or navitoclax (f). g, h After HCT116 WT and DKO cells were treated with TNFα+CHX (g) or navitoclax (h) for indicated time, representative microscopic images were taken. Scale bar in this and subsequent figures, 100 μm. i, j After TNFα+CHX (i) or navitoclax (j) treatment, HCT116 cells were collected at the indicated time points and stained with FITC-Annexin V and PI. The percentages of single PI positive, single FITC-Annexin V positive, and FITC-Annexin V/PI double positive HCT116 (WT or DKO) were detected by flow cytometry.

Fig. 2. Either BAK or BAX knockdown decreases TNFα+CHX or navitoclax-induced pyroptosis.

a Efficiency of BAK and/or BAX knockdown was detected by immunoblotting. b–e After BAK siRNA, BAX siRNA, BAK siRNA+BAX siRNA, or negative control siRNA were transfected into HCT116 cells by Lipofectamine RNAiMAX, cells were reseeded into 24-well plate followed by treatment of TNFα+CHX (b, d) or navitoclax (c, e) for 24 h. Culture supernatants were collected to measure the percentage of LDH release (b, c) and phase contrast images were taken (d, e) at the indicated time points.

Fig. 3. Caspase-3 is required for TNFα+CHX and navitoclax-induced pyroptosis.

a, b Immunoblottings of GSDME, caspases, and cleaved caspases in HCT116 WT and DKO cells treated with TNFα+CHX (a) or navitoclax (b) in the absence or presence of Q-VD-OPh at the indicated time points were performed. c, d The percentage of LDH release in the culture supernatants from HCT116 WT and DKO was measured after TNFα+CHX (c) or navitoclax (d) treatments in the absence or presence of Q-VD-OPh at the indicated time points. e, f After HCT116 WT and DKO cells were treated with TNFα+CHX (e) or navitoclax (f) in the absence or presence of Q-VD-OPh, the percentages of single PI positive, single FITC-Annexin V positive, and FITC-Annexin V/PI double positive cells were detected by flow cytometry at the indicated time points. g Efficiency of caspase-3 knockdown was detected by immunoblotting. h, i After HCT116 WT cells were transfected with caspase-3 siRNAs followed by TNFα+CHX (h) or navitoclax (i) treatments, culture supernatants were collected to measure the percentage of LDH release.

Fig. 4. GSDME knockdown decreases pyroptosis induced by TNFα+CHX or navitoclax.

a Efficiency of GSDME knockdown was detected by immunoblotting. b–e After HCT116 cells were transfected with GSDME siRNAs and negative control, cells were reseeded and treated with TNFα + CHX (b, d) or navitoclax (c, e) for the indicated time. The cells were collected for immunoblotting analysis (b, c) and culture supernatants were collected to detect the percentage of LDH release (d, e). f–i After HCT116 cells were transfected with GSDME siRNAs and negative control siRNA, cells were reseeded and treated with TNFα+CHX (f, g) or navitoclax (h, i). Phase contrast images were taken at the indicated time points (f, g) and the percentage of pyroptotic cells was calculated (h, i).

Fig. 5. GSDME is modified during anti-cancer drug-induced pyroptosis.

a, b After Hela cells were treated with TNFα+CHX (a) or actinomycin D (b) for the indicated time, the cells were subjected to immunoblotting. c, d TNFα+CHX treated Ovcar3 and HeyA8 cells for the indicated time and then harvested for immunoblotting. e The potential palmitoylation sites were predicted by CSS-Palm 4.0. f Sequence alignment of the potential palmitoylation site of GSDME from Danio rerio to Homo sapiens. Asterisk represents the conserved Cysteine residues were highlighted. g, h After Hela cells were pre-incubated with broad-spectrum palmitoylation inhibitors 2-BP followed by the treatment of TNFα+CHX for the indicated time, the cells were subjected to immunoblotting (g). Numbers indicated the ratio of optical density of shifted GSDME-C band to the unshifted GSDME-C band were obtained from four independent assays by using ImageJ software (h).

Fig. 6. 2-BP treatment inhibited TNFα+CHX induced pyroptosis.

a After 2-BP and TNFα+CHX treatment, Hela cells were collected at the indicated time points and stained with FITC-Annexin V and PI. The percentages of single PI positive, single FITC-Annexin V positive and FITC-Annexin V/PI double positive cells were detected by flow cytometry. b, c After 2-BP and TNFα+CHX treatment for the indicated time, Hela cells were subjected to Hoechst 33342 and PI double staining. Fluorescent microscopic images were taken at the indicated time points (c) and the ratio of PI positive cells were determined by Image J software (b). d After Hela cells were treated with 2-BP and TNFα+CHX for the indicated time, the culture supernatants were collected to measure the percentage of LDH release. e, f After Hela cells were transfected with GSDME WT or C407A/C408A mutant followed by the treatment of TNFα+CHX for the indicated time, cells and supernatants were collected for immunoblotting with anti-GSDME and anti-β-actin antibodies (e) and LDH detection (f). g 24 h after 3× Flag-tagged GSDME-N and WT or mutated S-tagged GSDME-C were co-transfected, Hela cells were treated with DMSO or 2-BP for 6 h, then cells were harvested for co-immunoprecipitations by anti-Flag antibody. The inputs were also subjected to immnuoblotting to show the expression levels. Asterisk represents unspecific band.

Fig. 7. GSDME interacts with ZDHHCs.

a, b After 3×Flag-tagged GSDME and indicated HA-tagged ZDHHCs were co-transfected into 293T cells, co-immunoprecipitations were performed using an anti-Flag antibody (a) or an anti-HA antibody (b). The inputs were also subjected to immunoblotting to show the expression levels. c The expression of GSDME and different ZDHHCs in CESC and CRC were summarized based on the immunohistochemistry results from the Human Protein Atlas. d Venn diagram was performed to show the expression patterns of different ZDHHCs in CESC and CRC, including expression level and subcellular distributions. e 24 h after Hela cells transfected with the indicated ZDHHC plasmids and then cells were incubated with actinomycin D for the indicated time. The cells were collected for immunoblotting. Numbers indicated the ratio of optical density of shifted GSDME-C band to the unshifted GSDME-C band. Arrows indicated the expression of different ZDHHC proteins.

Fig. 8. The model of chemotherapy-induced pyroptosis was indicated.

MOMP pathway inducers, such as TNFα+CHX, navitoclax and etoposide can activate BAK/BAX to permeabilize the MOM and release cytochrome c into the cytosol. Cytochrome c then activates caspase-9 and subsequent caspase-3, which will cause cell apoptosis or pyroptosis. In pyrototic pathway, GSDME is cleaved by caspase-3 to gnenrate GSDME-N and GSDME-C, where GSDME-N could directly oligomerize and cause plasma membrane lysis. Palmitoylation inhibitors inhibit GSDME-C palmitoylation, therefore inhibit its dissociation from GSDME-N and subsequent pyroptotic characteristics, including LDH release and the formation of plasma membrane bubbles.